WO1997018321A1 - Production photosynthetique de proteines de recombinaison marqueespar des isotopes stables - Google Patents

Production photosynthetique de proteines de recombinaison marqueespar des isotopes stables Download PDF

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Publication number
WO1997018321A1
WO1997018321A1 PCT/US1996/018229 US9618229W WO9718321A1 WO 1997018321 A1 WO1997018321 A1 WO 1997018321A1 US 9618229 W US9618229 W US 9618229W WO 9718321 A1 WO9718321 A1 WO 9718321A1
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WO
WIPO (PCT)
Prior art keywords
alga
organism
foreign protein
stable
cell
Prior art date
Application number
PCT/US1996/018229
Other languages
English (en)
Inventor
F. C. Thomas Allnutt
Original Assignee
Martek Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Martek Corporation filed Critical Martek Corporation
Priority to AU77316/96A priority Critical patent/AU7731696A/en
Publication of WO1997018321A1 publication Critical patent/WO1997018321A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • a method of producing recombinant protein which is uniformly labeled with a stable isotope comprises the steps of: photosynthetically culturing a recombinant cell or organism which encodes a foreign protein in a culture medium which comprises a stable isotopically labelled substrate selected from the group consisting of a simple 15 N-containing compound, 13 CO 2 , and 2 H 2 O, to produce said foreign protein which is labeled with a stable isotope, harvesting said recombinant cell or organism from said culture medium, and isolating said foreign protein from said harvested recombinant cell or organism or from said culture medium
  • Another method for producing recombinant protein which is uniformly labeled with a stable isotope comprises the steps of autotrophically culturing a recombinant photosynthetic cell or organism which encodes a foreign protein in a culture medium which comprises a stable isotopically labelled substrate, to produce said foreign protein which is labeled with the stable isotope; harvesting said photosynthetic cell or organism, and isolating said foreign protein from said harvested photosynthetic cell or organism or from said culture medium
  • stable isotopically labelled proteins can be made very inexpensively by production in photosynthetic cells or organisms, e.g., algal cells.
  • the photosynthetic cells or organisms can be grown on simple inorganic substrates which are labeled with a stable isotope.
  • one or more of 15 N 2 , 1 CO 2 , or 2 H, O can be used in the culture medium of the algae to achieve proteins which are uniformly labeled in their nitrogen, carbon or hydrogen atoms.
  • l 5 N, C, or 2 H may be used, as well as other stable isotopes, such as 18 0, 33 S, 34 S, 58 Fe, "Fe, 54 Fe, 7 ⁇ Zn, 67 Zn, 74 Se, 76 Se, 77 Se, 78 Se, 82 Se, 29 Si, 30 Si.
  • the isotope is oxygen, iron, or silicon
  • the photosynthetic cells or organisms are cultured autotrophically.
  • the gene encoding the desired foreign protein for labelling can be cloned into a vector according to techniques which are known in the art.
  • Foreign proteins according to the invention include any which are not found in nature in the particular organism being used.
  • the vector can be self-replicating in the photosynthetic cell or organism or it can incorporate into the genome
  • the gene-loaded vector can then be introduced into the target photosynthetic cell or organism via standard transformation procedures. These include natural uptake, conjugation, microprojectile bombardment, eiectroporation, and physical disruption by particulates such as glass beads or silicon whiskers.
  • Transformed cells can be plated on selective medium to select specific clones containing the desired genes. Clones which produce high amounts of the desired protein can also be selected or screened.
  • the desired foreign protein can be isolated from the harvested biomass or from the culture medium, if the protein is secreted from the host cell into the culture medium. Purification can be accomplished using affinity chromatography, or other suitable protein separation methods, as are known in the art.
  • Host strains for the desired recombinant protein can be any photosynthetic cell or organism known in the art. These include cyanobacteria and eukaryotic algae The cyanobacteria (blue-green algae) are prokaryotic organisms that have similar characteristics to other gram-negative bacteria.
  • the eukaryotic algae include the green as well as the brown algae, both of which can be used as the host cells for the recombinant protein
  • diatoms and other chrysophytes can be used, as well as dinoflagellates, red algae, cryptomonads, and euglenoids
  • Other photosynthetic bacteria can be used as well, including non-oxygenic purple sulfur and non-sulfur bacteria and nitrogen-fixing bacteria Single cell culture of higher plants may also be used
  • the recombinant foreign protein can be targeted for deposition in specific cells that are always anaerobic (the heterocysts), the cytoplasm, the periplasmic space, or the external medium
  • Specific vectors for incorporation of the gene into either the chloroplast or nuclear genomes can be used, or autonomously replicating vectors can be used
  • the gene for the desired foreign protein can be expressed either constitutively or by induction
  • vectors having sites of insertion downstream from suitable promoters for such control can be used, as is appropriate
  • genes for toxic proteins are introduced to a cell in a repressed state, induction is required for expression, but typically only after sufficient biomass is achieved in the culture
  • genes encoding toxic proteins can be targeted for expression in specialized cells or organelles which will effectively sequester the protein Desired proteins can be expressed in the cytoplasm, be directed to specific sites in the cell by leader sequences, or be directed for export from the cell Any such techniques as are known in the art can be used After isolation ofthe stable isotopically labeled protein

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  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Botany (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé peu coûteux permettant de produire des protéines de recombinaison marquées uniformément par des isotopes stables, faisant appel à des cellules ou à des organismes photosynthétiques tels que des algues qui se sont développées sur de simples substrats inorganiques qui sont marqués avec des isotopes stables.
PCT/US1996/018229 1995-11-15 1996-11-14 Production photosynthetique de proteines de recombinaison marqueespar des isotopes stables WO1997018321A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU77316/96A AU7731696A (en) 1995-11-15 1996-11-14 Photosynthetic production of stable isotopically labeled recombinant proteins

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US679295P 1995-11-15 1995-11-15
US60/006,792 1995-11-15

Publications (1)

Publication Number Publication Date
WO1997018321A1 true WO1997018321A1 (fr) 1997-05-22

Family

ID=21722612

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/018229 WO1997018321A1 (fr) 1995-11-15 1996-11-14 Production photosynthetique de proteines de recombinaison marqueespar des isotopes stables

Country Status (2)

Country Link
AU (1) AU7731696A (fr)
WO (1) WO1997018321A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2820758A1 (fr) * 2001-02-09 2002-08-16 Univ Pasteur Procede pour produire des proteines recombinantes, marquees par au moins un isotope
EP1548116A1 (fr) * 2002-09-30 2005-06-29 Ajinomoto Co., Inc. Procede de production d'une proteine marquee par un isotope stable
AT501629A1 (de) * 2005-04-05 2006-10-15 Erber Ag Herstellung von hochgradig isotopenmarkierten, sekundären, mikrobiellen stoffwechselprodukten sowie stoffwechselprodukte

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990015525A1 (fr) * 1989-06-14 1990-12-27 Martek Corporation Milieux destines a la croissance des cellules et procede servant a les produire
WO1991018105A1 (fr) * 1990-05-21 1991-11-28 Martek Corporation Composes marques utilises pour des tests diagnostiques de l'haleine
US5270175A (en) * 1991-07-12 1993-12-14 Dna Plant Technology Corporation Methods and compositions for producing metabolic products for algae
WO1994018339A1 (fr) * 1993-02-05 1994-08-18 Martek Biosciences Corporation Compositions et procedes de determination de structures proteiniques

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990015525A1 (fr) * 1989-06-14 1990-12-27 Martek Corporation Milieux destines a la croissance des cellules et procede servant a les produire
WO1991018105A1 (fr) * 1990-05-21 1991-11-28 Martek Corporation Composes marques utilises pour des tests diagnostiques de l'haleine
US5270175A (en) * 1991-07-12 1993-12-14 Dna Plant Technology Corporation Methods and compositions for producing metabolic products for algae
WO1994018339A1 (fr) * 1993-02-05 1994-08-18 Martek Biosciences Corporation Compositions et procedes de determination de structures proteiniques

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
COX, J., ET AL .: "STABLE-ISOTOPE-LABELED BIOCHEMICALS FROM MICROALGAE", TRENDS IN BIOTECHNOLOGY, vol. 6, 1988, pages 279 - 282, XP002026962 *
SODE, K., ET AL .: "FOREIGN GENE EXPRESSION IN MARINE CYANOBACTERIA UNDER PSEUDO-CONTINOUS CULTURE", JOURNAL OF BIOTECHNOLOGY, vol. 33, 1994, pages 243 - 248, XP002026961 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2820758A1 (fr) * 2001-02-09 2002-08-16 Univ Pasteur Procede pour produire des proteines recombinantes, marquees par au moins un isotope
WO2002064811A2 (fr) * 2001-02-09 2002-08-22 Universite Louis Pasteur Procede pour produire des proteines recombinantes, marquees par au moins un isotope
WO2002064811A3 (fr) * 2001-02-09 2003-03-20 Univ Pasteur Procede pour produire des proteines recombinantes, marquees par au moins un isotope
EP1548116A1 (fr) * 2002-09-30 2005-06-29 Ajinomoto Co., Inc. Procede de production d'une proteine marquee par un isotope stable
EP1548116A4 (fr) * 2002-09-30 2006-04-05 Ajinomoto Kk Procede de production d'une proteine marquee par un isotope stable
AT501629A1 (de) * 2005-04-05 2006-10-15 Erber Ag Herstellung von hochgradig isotopenmarkierten, sekundären, mikrobiellen stoffwechselprodukten sowie stoffwechselprodukte
AT501629B1 (de) * 2005-04-05 2007-10-15 Erber Ag Herstellung von hochgradig isotopenmarkierten, sekundären, mikrobiellen stoffwechselprodukten sowie stoffwechselprodukte

Also Published As

Publication number Publication date
AU7731696A (en) 1997-06-05

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